**6. Biological control:** *Bacillus thuringiensis*

Within the biological control market, biopesticides based on *Bacillus thuringiensis* are the most used worldwide due to their toxicity towards a wide range of pest insects from different orders and harmlessness to humans [32].

The insecticidal activity of most *B. thuringiensis* subspecies is due to their producing a cytoplasmic inclusion called δ-endotoxin, which is synthesized during the sporulation process [33]. The δ-endotoxins of the two *B. thuringiensis* subspecies *kurstaki* and *aizawai* are insecticidal against *L. botrana* larvae. This insecticidal action occurs when spores and endotoxins are ingested by the larvae, and then solubilized and turned into active toxins with lower molecular mass by insect proteases in the alkaline pH of larvae midgut. Active toxins bond to specific receptors and induce pore formation in the membrane of intestinal cells, causing membrane integrity loss and cellular lysis that allows bacteria to enter the hemocoel (insect circulatory system), finally leading to larval death due to starvation and sepsis [34]. *L. botrana* larval stage 1 is the most susceptible to δ-endotoxin action, so it is recommended to monitor grape bunches and apply this strategy to eggs in the black head development stage. In this way, emerging L1 larvae will have direct contact with the biopesticide.

The lethality of δ-endotoxins from *Bacillus thuringiensis* groups Cry1, Cry2 and Cry9 which presented activity against Lepidopterae was evaluated on L1 stage *L. botrana* larvae [35]. The toxins with the greatest insecticidal activity were Cry9Ca, Cry2Ab and Cry1Ab, with LC50 values of 0.09, 0.1 and 1.4 μg/ml, respectively. Cry9Ca and Cry1Ab do not share affinity with the same receptor, so combining both δ-endotoxins together with *B. thuringiensis* would allow for better control of L1 stage *L. botrana* larvae [35].

#### **7. Biological control: entomopathogenic fungi**

Entomopathogenic fungi (EPF) are microorganisms able to infect and naturally control arthropod populations, allowing them to be used as an alternative to chemical insecticides for pest control. In the microbial pest killer market, around

80% of available EPF products are based on species from the *Metarhizium* and *Beauveria* genera, since both have a wide range of hosts and are easy to massproduce [36]. *Metarhizium* and *Beauveria* include different species which over time have expanded, due to new types being isolated worldwide and the use of molecular techniques which allow for conclusive and certain identification.

EPF form complex relations with plants, apart from naturally controlling arthropod populations. Studies have shown that EPF species *M. robertsii* and *B. bassiana* provide plants part of the nitrogen which they absorb during insect parasitization [37, 38], promoting plant growth [39]. *Beauveria bassiana* has also been shown to act as an endophyte (colonizing plant interiors) in around 25 plant species, contributing to control of pests and phytopathogenic fungi [38, 40, 41]. It colonizes leaves, buds and roots, allowing plants to be more resistant to insect attacks [38, 42].

The action mechanism developed by EPF to parasitize insects requires EPF to differentiate into morphologically different cellular structures: conidium, germ tube, appressorium, hypha and blastospores. These structures participate in the insect infection and parasitizing process: conidia adhesion to the host cuticle (**Figure 8A**), formation and differentiation of the germinal tube in a structure called appressorium along with its penetration inside the insect cuticle (**Figure 8B**). Hemocoel colonization by blastospores (**Figure 8C**). Emergence of EPF hyphae from inside the insect and EPF sporulation on the corpse (**Figure 8D**), thereby promoting conidia dispersion and the start of new infections.

Although the action mechanism of EPF is known and interest in adopting biological pest control strategies is high, there are few scientific studies which have evaluated EPF effectiveness on *L. botrana* in field conditions. To this end, the study by Cozzi et al. [1] determined the lethality of 6 EPF isolates in an *in vitro* test on *L. botrana* larvae. The best strain, *B. bassiana* ITM 1559, showed a mortality rate of 55% of individuals of this pest. Furthermore, in field tests the incidence of bunches harmed by *L. botrana* larvae was significantly reduced via treatment with this strain, by comparison with the untreated control. In the study by Altimira et al. [19] 100%

#### **Figure 8.**

*Infection and development cycle of entomopathogenic fungus (EPF) on an insect pupa. Panel A: Conidium adhesion; panel B: Spore germination; panel C: Appressorium differentiation and cuticle penetration; panel D: Hemocoel colonization; panel E: Hyphae emergence and sporulation; panel F: Strata which EPF must cross to colonize the hemolymph.*

*Integrated Pest Management of* Lobesia botrana *with Microorganism in Vineyards… DOI: http://dx.doi.org/10.5772/intechopen.99153*

effectiveness was obtained against un-cocooned *L. botrana* pupae via using a wettable powder formulation of the strain *B. pseudobassiana* RGM 1747. This field test was done with a controlled infestation of *L. botrana* in ´Red Globe' *V. vinifera* during autumn (average temperature 9.1°C). In natural infestation trials, an effectiveness rate of 51% was achieved in different *V. vinífera* varieties with an average temperature of 8.4°C. During this period, the adhesion, germination and colonization of *B. pseudobassiana* in cocooned pupae was achieved, demonstrating its effectiveness in climate conditions with low temperatures, rain and high humidity present in this time of year in the Metropolitan Region of Chile [19]. Subsequently, Tapia [43] achieved 80% effectiveness with the inverse emulsion formula of the *M. robertsii* RGM 678 strain against *L. botrana* pupae in field tests, along with achieving a significantly lower percentage of male *L. botrana* captures compared to the control treatment.
